Computational mutagenesis reveals the role of active-site tyrosine in stabilising a boat conformatión for the substrate: QM/MM molecular dynamics studies of wild-type and mutant xylanases

摘要

Molecular dynamics simulations have been performed for non-covalent complexes of phenylb-xylobioside with the retaining endo-b-1,4-xylanase from B. circulans (BCX) and its Tyr69Phe mutantusing a hybrid QM/MM methodology. A trajectory initiated for the wild-type enzyme–substratecomplex with the proximal xylose ring bound at the –1 subsite (adjacent to the scissile glycosidic bond)in the 4C1 chair conformation shows spontaneous transformation to the 2,5B boat conformation, andpotential of mean force calculations indicate that the boat is ~30 kJ mol-1 lower in free energy than thechair. Analogous simulations for the mutant lacking one oxygen atom confirm the key role of Tyr69 instabilizing the boat in preference to the 4C1 chair conformation, with a relative free energy difference ofabout 20 kJ mol-1, by donating a hydrogen bond to the endocyclic oxygen of the proximal xylose ring.QM/MM MD simulations for phenyl b-xyloside in water, with and without a propionate/propionicacid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the 4C1 chair to bestable, although a hydrogen bond between the OH group at C2 of xylose and the propionate moietyseems to provide some stabilization for the 2,5B conformation